Methods of modulating the expression of genes using ultraspiracle receptor

ABSTRACT

In accordance with the present invention, it has been discovered that various members of the steroid/thyroid superfamily of receptors can interact with the insect-derived ultraspiracle receptor, to form multimeric species. Accordingly, the interaction of at least one member of the steroid/thyroid superfamily of receptors with at least the dimerization domain of the ultraspiracle receptor modulates the ability of said member of the steroid/thyroid superfamily of receptors to trans-activate transcription of genes maintained under hormone expression control in the presence of the cognate ligand for said member of the superfamily.

RELATED APPLICATIONS

This application is a divisional of application U.S. Ser. No.07/907,908, filed Jul. 2, 1992, now abandoned, which is a continuationof application U.S. Ser. No. 07/497,935, filed Mar. 22, 1990, nowabandoned, the entire contents of which are hereby incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to interactions between members of thesteroid/thyroid superfamily of receptor proteins, novel combinations ofvarious members of the steroid/thyroid superfamily of receptor proteins,and methods of using such combinations.

BACKGROUND OF THE INVENTION

Transcriptional regulation of development and homeostasis in complexeukaryotes, including humans and other mammals, birds, fish, insects,and the like, is controlled by a wide variety of regulatory substances,including steroid and thyroid hormones. These hormones exert potenteffects on development and differentiation of phylogenetically diverseorganisms. The effects of hormones are mediated by interaction withspecific, high affinity binding proteins referred to as receptors.

A number of receptor proteins are known, each specific for steroidhormones [e.g., estrogens (estrogen receptor), progesterones(progesterone receptor), glucocorticoid (glucocorticoid receptor),androgens (androgen receptor), aldosterones (mineralocorticoidreceptor), vitamin D (vitamin D receptor)], retinoids (e.g., retinoicacid receptor) or thyroid hormones (e.g., thyroid hormone receptor).Receptor proteins have been found to be distributed throughout the cellpopulation of complex eukaryotes in a tissue specific fashion.

Molecular cloning studies have made it possible to demonstrate thatreceptors for steroid, retinoid and thyroid hormones are allstructurally related and comprise a superfamily of regulatory proteins.These regulatory proteins are capable of modulating specific geneexpression in response to hormone stimulation by binding directly tocis-acting elements.

It is known that steroid or thyroid hormones, protected forms thereof,or metabolites thereof, enter cells and bind to the correspondingspecific receptor protein, initiating an allosteric alteration of theprotein. As a result of this alteration, the complex of receptor andhormone (or metabolite thereof) is capable of binding with high affinityto certain specific sites on chromatin. One of the primary effects ofsteroid and thyroid hormones is an increase in transcription of a subsetof genes in specific cell types.

A number of transcriptional control units which are responsive tomembers of the steroid/thyroid superfamily of receptors have beenidentified. These include the mouse mammary tumor virus 5′-long terminalrepeat (MTV LTR), responsive to glucocorticoid, aldosterone and androgenhormones; the transcriptional control units for mammalian growth hormonegenes, responsive to glucocorticoids, estrogens and thyroid hormones;the transcriptional control units for mammalian prolactin genes andprogesterone receptor genes, responsive to estrogens; thetranscriptional control units for avian ovalbumin genes, responsive toprogesterones; mammalian metallothionein gene transcriptional controlunits, responsive to glucocorticoids; and mammalian hepaticα_(2u)-globulin gene transcriptional control units, responsive toandrogens, estrogens, thyroid hormones, and glucocorticoids.

A major obstacle to further understanding and more widespread use of thevarious members of the steroid/thyroid superfamily of hormone receptorshas been a lack of awareness of the possible interactions of variousmembers of the steroid/thyroid superfamily of hormone receptors, and anunderstanding of the implications of such interactions on the ability ofmembers of the steroid/thyroid superfamily of hormone receptors to exerttranscriptional regulation of various physiological processes.

DNA binding studies on the glucocorticoid receptor (GR) and the estrogenreceptor (ER) have indicated that these receptors bind to their hormoneresponse elements (HREs) as homodimeric complexes [see, for example,Kumar and Chambon in Cell 55:145-156 (1988) and Tsi et al., in Cell55:361-369 (1988)). However, recent biochemical analysis has revealedthat some other receptors (including retinoic acid receptor (RAR),thyroid hormone receptor (TR), and the vitamin D receptor (VDR)) can notefficiently bind to cognate response elements as homodimers, but ratherrequire additional factors present in cell nuclear extracts to achievehigh affinity DNA binding (see, for example, Murray and Towle in Mol.Endocrinol. 3:1434-1442 (1989), Glass et al., in Cell 63:729-738 (1990),Liao et al., in Proc. Natl. Acad. Sci. USA 87:9751-9755 (1990), and Yanget al., in Proc. Natl. Acad. Sci. USA 88:3559-3563 (1991)].

Several recent reports have identified members of the retinoid Xreceptor family (RXR; see, for example, Mangelsdorf et al., in Nature345:224-229 (1990) and Gene Dev. 6:329-344 (1992), and Leid et al., inCell 68:377-395 (1992)) as factors that can interact with RAR andpotentiate DNA binding by forming a novel RAR/RXR heterodimer [see, forexample, Yu et al., in Cell 67:1251-1266 (1991), Kliewer et al., inNature 355:446-449 (1992), Leid et al., supra, and Zhang et al., inNature 355:441-446 (1992)). Interestingly, RAR is not the only receptorwith which RXR can interact. In fact, RXR has been found to be capableof heterodimerizing with several other members of the nuclear receptorsuperfamily, including VDR, TR (see Kliewer, et al., supra) andperoxisome proliferator-activated receptor (PPAR; see, for example,Issemann and Green in Nature 347:645-650 (1990)).

Although the physiological significance of these interactions remains tobe definitively determined, the capability of nuclear receptors toheterodimerize suggests the existence of an elaborate network throughwhich distinct nuclear hormone receptor classes are capable ofmodulating each other's activity. In addition, the possible existence ofother factors that can potentially interact with members of thesteroid/thyroid superfamily and potentiate DNA binding by forming novelheteromeric species remains to be determined.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have discovered thatvarious members of the steroid/thyroid superfamily of receptors cancombine with the insect derived ultraspiracle receptor (or functionalfragments thereof comprising at least the dimerization domain thereof)to form a multimeric complex receptor. Accordingly, the combination of afirst receptor species with the ultraspiracle receptor (or a truncatedform thereof comprising at least the dimerization domain thereof) iscapable of modulating the ability of the first receptor species totrans-activate transcription of genes maintained under steroid hormoneor hormone-like expression control in the presence of cognate ligand forsaid first receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a comparison of amino acid identity for various domainsof invention ultraspiracle receptor (usp) in comparison with previouslyidentified receptors human RXR-alpha (hRXRα), human retinoic acidreceptor-alpha (hRARα) and human glucocorticoid receptor (hGR).

FIGS. 2A-3 present the % conversion of substrate by chloramphenicolacetyltransferase (CAT) as a result of cotransfection of mammalian (CV1)cells with ecdysone receptor (EcR) encoding vector and/or ultraspiraclereceptor (usp) encoding vector along with CAT reporter vector whichcontains an ecdysone response element (EcRE).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided multimericreceptor species which belong to the steroid/thyroid superfamily ofreceptors, comprising at least one member of the steroid/thyroidsuperfamily of receptors, and the ultraspiracle receptor.

As employed herein, the term “dimerization domain(s)” of a member of thesteroid/thyroid superfamily of receptors refers to that portion (orportions) of the receptor which is involved in the formation of a givenmultimeric complex receptor. Dimerization domain(s) typically compriseat least a portion of the carboxy-terminal portion of the receptor(i.e., the carboxy-terminal portion of a receptor with respect to theDNA-binding domain thereof) and/or at least a portion of the DNA bindingdomain itself. Multiple domains of a given receptor can act in concertas well as independently.

Combinations contemplated by the present invention can broadly bereferred to as “multimeric species”, which is intended to embrace all ofthe various oligomeric forms in which members of the steroid/thyroidsuperfamily of receptors (including fragments thereof comprising thedimerization domains thereof) are capable of associating with at leastthe dimerization domain of the ultraspiracle receptor. Thus, referenceto “combinations” of steroid receptors or “multimeric” forms of steroidreceptors with at least the dimerization domain of the ultraspiraclereceptor includes heterodimeric combinations of one member of thesteroid/thyroid superfamily of receptors (including fragments thereofcomprising the dimerization domain thereof) with at least thedimerization domain of the ultraspiracle receptor, heterotrimericcombinations of one or two members of the steroid/thyroid superfamily ofreceptors (including fragments thereof comprising the dimerizationdomains thereof) with at least the dimerization domain of theultraspiracle receptor, heterotetrameric combinations of one, two orthree members of the steroid/thyroid superfamily of receptors (includingfragments thereof comprising the dimerization domains thereof) with atleast the dimerization domain of the ultraspiracle receptor, and thelike.

As employed herein, the term “ultraspiracle receptor” refers to a novelinvertebrate polypeptide which has a DNA binding domain of about 66amino acids with at least 9 Cys residues, more than about 75% amino acididentity in comparison with the DNA binding domain of hRXR-alpha (seeMangelsdorf et al., 1990, supra), less than about 60% amino acididentity in comparison with the DNA binding domain of hGR, and less thanabout 60% amino acid identify in comparison with the DNA binding domainof hRARα. Invention polypeptide can be further characterized by havingless than 50% (but typically greater than 40%) amino acid identity incomparison with the ligand binding domain of hRXR-alpha, but less than25% amino acid identity in comparison with the ligand binding domains ofeither hGR or hRARα. A sequence comparison of amino acid identitybetween invention receptor and several other receptors is presented inFIG. 1.

The deduced amino acid sequence for the ultraspiracle receptor ispresented in SEQ ID NO:2[see also, Oro et al., in Nature 347:298-301(1990)]. Also contemplated within the scope of the present invention arepeptides comprising a DNA binding domain with substantially the samesequence as that of amino acids 104-169 shown in SEQ ID NO:2 (i.e., theDNA binding domain of the ultraspiracle receptor). As employed herein,the term “substantially the same amino acid sequence” refers to aminoacid sequences having at least about 80% identity with respect to thereference amino acid sequence, and retaining comparable functional andbiological properties characteristic of the protein encoded by thereference amino acid sequence. Preferably, proteins having“substantially the same amino acid sequence” will have at least about90% amino acid identity with respect to the reference amino acidsequence; with greater than about 95% amino acid sequence identity beingespecially preferred. Also contemplated within the scope of the presentinvention are polypeptides having substantially the same sequence asthat of amino acids 1-513 shown in SEQ ID NO:2. A presently preferredpolypeptide of the invention is the polypeptide encoded by vector pXR2C8[see Oro et al., supra].

As employed herein, the phrase “members of the steroid/thyroidsuperfamily of receptors” refers to all of the various isoforms ofhormone binding proteins that operate as ligand-dependent transcriptionfactors, including members of the steroid/thyroid superfamily ofreceptors for which specific ligands have not yet been identified(referred to hereinafter as “orphan receptors”). Each such protein hasthe intrinsic ability to bind to a specific DNA sequence (i.e.,regulatory sequence) associated with the target gene. Thetranscriptional activity of the gene is modulated by the presence orabsence of the cognate hormone (ligand) as a result of binding of ligandto receptor, enabling interaction of receptor with the regulatorysequence.

The DNA-binding domains of all members of this superfamily of receptorsare related, consisting of 66-68 amino acid residues, and possessingabout 20 invariant amino acid residues, including nine cysteines. Amember of the superfamily can be characterized as a protein whichcontains these diagnostic amino acid residues, which are part of theDNA-binding domain of such known steroid receptors as the humanglucocorticoid receptor (amino acids 421-486), the estrogen receptor(amino acids 185-250), the mineralocorticoid receptor (amino acids603-668), the human retinoic acid receptor (amino acids 88-153), and thelike. The highly conserved amino acids of the DNA-binding domain ofmembers of the superfamily are as follows:

                         (SEQ ID No 3)Cys - X - X - Cys - X - X - Asp* - X -Ala* - X - Gly* - X - Tyr* - X - X -X - X - Cys - X - X - Cys - Lys* - X -Phe - Phe - X - Arg* - X - X - X - X -X - X - X - X - X - (X - X -) Cys - X -X - X - X - X - (X - X - X -) Cys - X -X - X - Lys - X - X - Arg - X - X - Cys - X - X - Cys - Arg* - X - X -Lys* - Cys - X - X - X - Gly* - Met;

wherein X designates non-conserved amino acids within the DNA-bindingdomain; the amino acid residues denoted with an asterisk are residuesthat are almost universally conserved, but for which variations havebeen found in some identified hormone receptors; and the residuesenclosed in parenthesis are optional residues (thus, the DNA-bindingdomain is a minimum of 66 amino acids in length, but can contain severaladditional residues).

Exemplary members of the steroid/thyroid superfamily of receptors(including the various isoforms thereof) include steroid receptors suchas glucocorticoid receptor, mineralocorticoid receptor, progesteronereceptor, androgen receptor, vitamin D₃ receptor, and the like; plusretinoid receptors, such as the various isoforms of RAR (e.g., RARα,RARβ, or RARλ), the various isoforms of RXR (e.g., RXRα, RXRβ, or RXRλ),and the like; thyroid receptors, such as TRα, TRβ, and the like; insectderived receptors such as the ecdysone receptor, and the like; as wellas other gene products which, by their structure and properties, areconsidered to be members of the superfamily, as defined hereinabove,including the various isoforms thereof (even though ligands thereforhave not yet been identified; such receptors are referred to as “orphanreceptors”). Examples of orphan receptors include HNF4 [see, forexample, Sladek et al., in Genes & Development 4:2353-2365 (1990)], theCOUP family of receptors (see, for example, Miyajima et al., in NucleicAcids Research 16:11057-11074 (1988), and Wang et al., in Nature340:163-166 (1989)], COUP-like receptors and COUP homologs, such asthose described by Mlodzik et al., in Cell 60:211-224 (1990) and Ladiaset al., in Science 251:561-565 (1991), various isoforms of peroxisomeproliferator-activated receptors (PPARs; see, for example, Issemann andGreen, supra), the insect derived knirps and knirps-related receptors,and the like.

The formation of multimeric receptor(s) can modulate the ability ofmember(s) of the steroid/thyroid superfamily of receptors totrans-activate transcription of genes maintained under expressioncontrol in the presence of ligand for said receptor. The actual effecton activation of transcription (i.e., enhancement or repression oftranscription activity) will vary depending on the receptor specieswhich are part of the multimeric receptor, as well as on the responseelement with which the multimeric species interacts. Thus, for example,formation of a heterodimer of the ecdysone receptor with theultraspiracle receptor promotes the ability of the ecdysone receptor toinduce trans-activation activity in the presence of an ecdysone responseelement (see, for example, SEQ ID NO:26).

In accordance with another embodiment of the present invention, there isprovided a method to modulate, in an expression system, thetranscription activation of a gene by a member of the steroid/thyroidsuperfamily of receptors in the presence of ligand therefor, wherein theexpression of said gene is maintained under the control of a hormoneresponse element, said method comprising:

exposing said system to at least the dimerization domain of theultraspiracle receptor, in an amount effective to form a multimericcomplex receptor with said member of the steroid/thyroid superfamily ofreceptors.

Exposure of said system to at least the dimerization domain of theultraspiracle receptor is accomplished by directly administeringultraspiracle receptor (or fragments thereof that allow modification ofthe receptor through the formation of heterodimeric receptor species) tosaid system, or by exposing said system to compound(s) and/orcondition(s) which induce expression of the ultraspiracle receptor (ordimerization domain thereof). The resulting multimeric receptor speciesis effective to modulate transcription activation of said gene.

As employed herein, the term “modulate” refers to the ability of a givenmultimeric complex receptor to either enhance or repress the inductionof transcription of a target gene by a given receptor, relative to suchability of said receptor in its uncomplexed state. The actual effect ofmultimerization on the transcription activity of a receptor will varydepending on the specific receptor species which are part of themultimeric complex receptor, and on the response element with which themultimeric complex receptor interacts. Thus, for example, formation of aheterodimer of the ecdysone receptor and the ultraspiracle receptorprovides enhanced trans-activation activity with respect to the abilityof the ecdysone receptor alone to promote trans-activation. Conversely,formation of a heterodimer of the ecdysone receptor and the dimerizationdomain of the ultraspiracle receptor should prevent the ability ofecdysone to promote trans-activation activity, since the resultingmultimeric complex receptor will have a reduced ability to bind DNA,relative to the ability of ecdysone-usp multimeric complex to bind DNA.

The term “ecdysone” is employed herein in its generic sense (inaccordance with common usage in the art), referring to compounds withthe appropriate biological activity, in analogy with the terms estrogenor progestin [see, for example, Cherbas et al., in Biosynthesis,metabolism and mode of action of invertebrate hormones (ed. J. Hoffmannand M. Porchet), p. 305-322; Springer-Verlag, Berlin].20-Hydroxyecdysone is the major naturally occurring ecdysone. Analogs ofthe naturally occurring ecdysones are also contemplated within the scopeof the present invention, such as for example, ponasterone A,26-iodoponasterone A, muristerone, inokosterone, 26-mesylinokosterone,and the like.

As employed herein, the phrase “hormone response element” refers toshort cis-acting sequences (i.e., having about 20 bp) that are requiredfor hormonal (or ligand) activation of transcription. The attachment ofthese elements to an otherwise hormone-nonresponsive gene causes thatgene to become hormone responsive. These sequences, commonly referred toas hormone response elements (or HREs), function in a position- andorientation-independent fashion. Unlike other enhancers, the activity ofHREs can be modulated by the presence or absence of ligand. See, forexample, Evans, Science 240:889-895 (1988), and the references citedtherein. In the present specification and claims, the term “hormoneresponse element” is used in a generic sense to mean and embody thefunctional characteristics implied by all terms used in the art todescribe these sequences.

Hormone response elements contemplated for use in the practice of thepresent invention include naturally occurring response elements as wellas modified forms thereof (see, for example, SEQ ID NOs:7, 12, 15, 25,26, 28 and 29), as well as synthetic response elements which can becomposed of two or more “half sites”, wherein each half site comprisesthe sequence

—RGBNNM—,

wherein

R is selected from A or G;

B is selected from G, C, or T;

each N is independently selected from A, T, C, or G; and

M is selected from A or C;

with the proviso that at least 4 nucleotides of said —RGBNNM— sequenceare identical with the nucleotides at corresponding positions of thesequence —AGGTCA—, or the half-sites of ecdysone response elements(EcREs) (see, for example, SEQ ID NOs:26, 28 and 29) and

wherein the nucleotide spacing between each of said half-sites falls inthe range of 0 up to 15 nucleotides, N.

When one of the half sites described above is incorporated into asynthetic response element in a direct repeat motif, and such half sitevaries by 2 nucleotides from the preferred sequence of —AGGTCA—, it ispreferred that the other half site of the response element be the sameas, or vary from the preferred sequence by no more than 1 nucleotide. Itis presently preferred that the 3′-half site (or downstream half site)of a pair of half sites vary from the preferred sequence by at most 1nucleotide.

When the above-described half sites are combined in direct repeatfashion (rather than as palindromic constructs), the resulting syntheticresponse elements are referred to as “DR-x”, wherein “DR” refers to thedirect repeat nature of the association between the half sites, and “x”indicates the number of spacer nucleotides between each half site.

Exemplary response elements useful in the practice of the presentinvention are derived from various combinations of half sites havingsequences selected from, for example, —AGGTCA—, —GGTTCA—, —GGGTTA—,—GGGTGA—, —AGGTGA—, —GGGTCA—, and the like.

The nucleotides employed in a non-zero spacer are independently selectedfrom C, T, G, or A.

Exemplary three nucleotide spacers include —AGG—, —ATG—, —ACG—, —CGA—,and the like. Exemplary four nucleotide spacers include —CAGG—, —GGGG—,—TTTC—, and the like. Exemplary five nucleotide spacers include —CCAGG—,—ACAGG—, —CCGAA—, —CTGAC—, —TTGAC—, and the like.

Exemplary response elements contemplated by the present inventioninclude the following DR-3 elements:

5′-AGGTCA-AGG-AGGTCA-3′ (SEQ ID No. 4), 5′-GGGTGA-ATG-AGGACA-3′ (SEQ IDNo. 5), 5′-GGGTGA-ACG-GGGGCA-3′ (SEQ ID No. 6), and5′-GGTTCA-CGA-GGTTCA-3′ (SEQ ID No. 7);

the following DR-4 elements:

5′-AGGTCA-CAGG-AGGTCA-3′ (SEQ ID No. 8), 5′-AGGTGA-CAGG-AGGTCA-3′ (SEQID No. 9), 5′-AGGTGA-CAGG-AGGACA-3′ (SEQ ID No. 10),5′-GGGTTA-GGGG-AGGACA-3′ (SEQ ID No. 11),

and

5′—GGGTCA—TTTC—AGGTCC—3′ (SEQ ID No.12); the following DR-5 elements:

5′-AGGTCA-CCAGG-AGGTCA-3′ (SEQ ID No. 13), 5′-AGGTGA-ACAGG-AGGTCA-3′(SEQ ID No. 14), 5′-GGTTCA-CCGAA-AGTTCA-3′ (SEQ ID No. 15),5′-GGTTCA-CCGAA-AGTTCA-3′ (SEQ ID No. 16), 5′-GGTTCA-CTGAC-AGGGCA-3′(SEQ ID No. 17), 5′-GGGTCA-TTCAG-AGTTCA-3′ (SEQ ID No. 18),5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCAGCTT-3′ (SEQ ID No. 19),5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATAGCTT-3′ (SEQ ID No. 20), and5′-AAGCTTAAG-GGTTCA-CCGAA-AGTTCA-CTCGCATATATTAGCTT-3′ (SEQ ID No. 21);

the ecdysone responsive elements set forth in SEQ ID NOs:26, 28 and 29,and the like.

Presently preferred response elements contemplated for use in thepractice of the present invention include:

5′-AGGTCA-AGG-AGGTCA-3′ (SEQ ID No. 4), 5′-AGGTCA-CAGG-AGGTCA-3′ (SEQ IDNo. 8), 5′-AGGTGA-CAGG-AGGTCA-3′ (SEQ ID No. 9),5′-AGGTCA-CCAGG-AGGTCA-3′ (SEQ ID No. 13), 5′-AGGTGA-ACAGG-AGGTCA-3′(SEQ ID No. 14), SEQ ID NOs:26, 28, 29, and the like.

These are especially preferred because they represent synthetic and/orinvertebrate sequences which have not been observed in vertebrates, andthus are applicable to a wide variety of reporter systems (i.e., the useof these response elements will not be limited due to any speciespreference based on the source of the sequence).

In accordance with yet another embodiment of the present invention,there is provided a method to modulate, in an expression system, thetranscription activation of a gene by a member of the steroid/thyroidsuperfamily of receptors in the presence of ligand therefor, and in thefurther presence of the ultraspiracle receptor, wherein the expressionof said gene is maintained under the control of a hormone responseelement, said method comprising:

exposing said system to compound(s) and/or condition(s) which preventassociation of said member with the ultraspiracle receptor or fragmentsthereof, in an amount effective to prevent said association.

Compound(s) and/or condition(s) which prevent association of said memberwith the ultraspiracle receptor include hormone-like compounds which actas agonists or antagonists for the ultraspiracle receptor, antibodiesraised against the dimerization domain of the ultraspiracle receptor,antibodies raised against the dimerization domain of said member,antisense sequence(s) based on sequence(s) complementary to known RNAencoding at least the dimerization domain of the ultraspiracle receptor,and the like. Amounts of agents effective to prevent such associationwill vary depending on the particular agents used and can be readilydetermined by those of skill in the art; typically falling in thesub-nanomolar up to micromolar range.

In accordance with still another embodiment of the present invention,there is provided a method for modulating the expression of an exogenousgene in a subject containing:

(i) a DNA construct encoding said exogenous gene under the control of asteroid or steroidlike hormone response element; wherein said responseelement is not normally present in the cells of said subject,

(ii) a receptor which is not normally present in the cells of saidsubject, wherein said receptor, in the presence of its associated ligandand the ultraspiracle receptor, binds to said steroid or steroid-likehormone response element, and

(iii) the ultraspiracle receptor;

said method comprising administering to said subject an effective amountof said associated ligand; wherein said ligand is not normally presentin the cells of said subject; and wherein said ligand is not toxic tosaid subject.

As employed herein, the term “exogenous” (or “foreign”) genes refers toboth wild type genes and therapeutic genes, which are introduced intothe subject in the form of DNA or RNA, either natural or synthetic. Thegene of interest can be introduced into target cells (for in vitroapplications), or the gene of interest can be introduced directly into asubject, or indirectly introduced by the transfer of transformed cellsinto a subject.

“Wild type” genes are those that are native to cells of a particulartype, but which may be undesirably overexpressed in these cells, or maynot be expressed in these cells in biologically significant levels.Thus, for example, while a synthetic or natural gene coding for humaninsulin would be exogenous genetic material to a yeast cell (since yeastcells do not naturally contain insulin genes), a human insulin geneinserted into a human skin fibroblast cell would be a wild type genewith respect to that cell since human skin fibroblasts contain thegenetic material encoding human insulin, although human skin fibroblastsdo not express human insulin in biologically significant levels.

Wild type genes contemplated for use in the practice of the presentinvention include genes which encode a gene product:

the substantial absence of which leads to the occurrence of a non-normalstate in said subject; or

a substantial excess of which leads to the occurrence of a non-normalstate in said subject;

and the like.

As employed herein, the phrase “therapeutic gene” refers to genes whichimpart a beneficial function to the host cell in which such gene isexpressed. Therapeutic genes are those that are not naturally found inhost cells. For example, a synthetic or natural gene coding forauthentic human insulin would be therapeutic when inserted into a skinfibroblast cell so as to be expressed in a host human, where the hosthuman is not otherwise capable of expressing functionally active humaninsulin in biologically significant levels.

Therapeutic genes contemplated for use in the practice of the presentinvention include genes which encode a gene product:

which is toxic to the cells in which it is expressed; or

which imparts a beneficial property to said subject (e.g., diseaseresistance, etc);

and the like.

Exogenous genetic material or genes useful in this embodiment of thepresent invention include genes that encode secretory proteins that canbe released from said cell; enzymes that can metabolize a substrate froma toxic form to a benign form, or from a benign form to a useful form;regulatory proteins; cell surface receptors; and the like. Such usefulgenes include, but are not limited to, genes that encode blood clottingfactors such as human factors VIII and IX; genes that encode hormonessuch as insulin, parathyroid hormone, luteinizing hormone releasingfactor (LHRH), alpha and beta seminal inhibins, and human growthhormone; genes that encode proteins such as enzymes, the absence ofwhich leads to the occurrence of an abnormal state in said subject;genes encoding cytokines or lymphokines such as interferons,granulocytic macrophage colony stimulating factor (GM-CSF), colonystimulating factor-1 (CSF-1), tumor necrosis factor (TNF), anderythropoietin (EPO); genes encoding inhibitor substances such asalpha,-antitrypsin; genes encoding substances that function as drugs,e.g., genes encoding the diphtheria and cholera toxins; and the like.

Hormone response elements contemplated for use in this embodiment of thepresent invention involving modulating the expression of an exogenousgene in a subject include any sequence responsive to the above-describedmultimeric complex receptors, such as insect response elements, and thelike. See, for example, SEQ ID NOs:26, 28 and 29.

Insect response elements contemplated for use in modulating theexpression of an exogenous gene in a subject according to the presentinvention include, for example, ecdysone response elements, and thelike.

Such response elements are operably linked to a suitable promoter forexpression of the target gene product. As used herein, the term“promoter” refers to a specific nucleotide sequence recognized by RNApolymerase, the enzyme that initiates RNA synthesis. This sequence isthe site at which transcription can be specifically initiated underproper conditions. When exogenous genes, operatively linked to asuitable promoter, are introduced into the cells of a suitable host, theexogenous genes are subject to expression control in the presence ofhormone or hormone-like compounds not normally present in the hostcells. Exemplary promoters include ΔMTV, ΔSV, ΔADH promoters, and thelike.

As employed herein, the phrase “receptor which is not normally presentin the cells of said subject” refers to receptors which are notendogenous to the host in which the invention process is being carriedout. Receptors which are not endogenous to the host include endogenousreceptors modified so as to be non-responsive to ligands which areendogenous to the host in which the invention process is being carriedout.

Receptor(s) not normally present in the cells of the subject andultraspiracle receptor (or fragments thereof) can be provided to saidsubject by direct introduction of the proteins themselves, byintroduction of RNA or DNA construct(s) encoding said receptors, byintroduction of cells harboring genes encoding said receptor and/orresponse element, and the like. This can be accomplished in a variety ofways, e.g., by microinjection, retroviral infection, electroporation,lipofection, and the like.

As employed herein, the phrase “associated ligand” refers to a substanceor compound which, inside a cell, binds to the receptor protein, therebycreating a ligand/receptor complex, which in turn can bind to anappropriate hormone response element. An associated ligand therefore isa compound which acts to modulate gene transcription for a genemaintained under the control of a hormone response element, and includescompounds such as hormones, growth substances, non-hormone substancesthat regulate growth, and the like. Ligands include steroid orsteroid-like hormones, retinoids, thyroid hormones, pharmaceuticallyactive compounds, and the like. Individual ligands may have the abilityto bind to multiple receptors.

In accordance with a still further embodiment of the present invention,there is provided a method of inducing the expression of an exogenousgene in a subject containing:

(i) a DNA construct encoding an exogenous gene product under the controlof a hormone response element; wherein said response element is notnormally present in the cells of said subject,

(ii) DNA encoding a receptor which is not normally present in the cellsof said subject, under the control of an inducible promoter; whereinsaid receptor, in the presence of its associated ligand and theultraspiracle receptor, binds to said hormone response element,

(iii) the ultraspiracle receptor, and

(iv) the associated ligand for said receptor;

said method comprising subjecting a subject to conditions suitable toinduce expression of said receptor.

In accordance with yet another embodiment of the present invention,there is provided a method of inducing expression of an exogenous geneproduct in a subject containing a DNA construct encoding said productunder the control of a hormone response element; wherein said responseelement is not normally present in the cells of said subject, saidmethod comprising introducing into said subject:

a receptor which is not normally present in the cells of said subject;wherein said receptor, in combination with its associated ligand and theultraspiracle receptor, binds to a hormone response element, activatingtranscription therefrom,

the ultraspiracle receptor, and

the associated ligand for said receptor which is not normally present inthe cells of said subject.

In accordance with this embodiment of the present invention, receptorcan be provided directly to said subject as the protein, or indirectlyby administering to said subject a second DNA construct encoding saidreceptor, or by administering to said subject cells harboring suchconstructs. When introduced as part of a second DNA construct,expression of said exogenous gene product and the receptor is preferablymaintained under the control of a tissue specific promoter.

In accordance with a further embodiment of the present invention, thereis provided a method for the expression of recombinant productsdetrimental to a host organism, said method comprising:

transforming suitable host cells with:

(i) a construct comprising a sequence encoding said recombinant productunder the control of a hormone response element;

wherein said response element is not normally present in the cells ofsaid host, and

(ii) DNA encoding a receptor not normally present in said host cells;

growing said host cells to the desired level in the substantial absenceof hormone(s) which, in combination with said receptor not normallypresent in the cells of said host and ultraspiracle receptor, is capableof binding to said hormone response element, and

inducing expression of said recombinant product by introducing into saidhost cells the ultraspiracle receptor and hormone(s) which, incombination with said receptor not normally present in the cells of saidhost, bind to said response element.

In one aspect of this embodiment of the invention, wherein host isemployed as an expression system for the production of a recombinantproduct which is toxic to the host, recombinant product is induced onlyafter cell growth (as opposed to protein expression conditions) hasproduced a desired density of cell mass. Thus, the desired level ofgrowth in accordance with this embodiment is a level which produces ahigh cell density, and thereafter expression of product is induced.Conditions suitable for cell growth (and for protein expression, whendesired) can be readily determined by those of skill in the art.

In another aspect of this embodiment of the. present invention, whereinthe host harbors a DNA construct as described above, expression of theconstruct to produce the detrimental product causes ablation of thecells harboring said construct. In this aspect, the desired level ofgrowth is that level appropriate to ensure the desired distribution ofcells harboring the inducible construct. Thus, expression will beinduced when it is desired to ablate such cells.

As used herein, “ablation” refers to removing or eliminating specificcell types in a culture of a cell population, or in a transgenic animalhost by means of a DNA construct that encodes a protein whose presenceis not per se toxic to the cells, but which can confer upon the cells atoxic potential due to the ability of the protein to control theexpression of substances that are or will become toxic to the cells.

The elimination of specific cell-type(s) or specific cell line(s) inaccordance with one aspect of the present invention produces a cellpopulation which is substantially free of cells which are not normallypresent in the wild-type cell population. The elimination of specificcell-type(s) or specific cell line(s), in accordance with another aspectof the present invention, produces a defined altered state in thetreated subject.

Cell(s) or cell line(s) contemplated to be eliminated in accordance withthe present invention can be a cell or cell line capable of providing adesirable component to a cell population, as an exogenous gene product;wherein the ability to eliminate said cell or cell line from said cellpopulation is desired, e.g., once said population achieves the abilityto produce sufficient quantities of such component as an endogenous geneproduct; or, the cell line to be eliminated can be a diseased cell lineor a cell line predisposed to a disease state.

Normal cell(s) or cell line(s) contemplated to be eliminated inaccordance with the present invention are cell(s) or cell line(s), theelimination of which would result in the creation of a defined alteredstate in the cell population.

In accordance with a still further embodiment of the present invention,there is provided a method to distinguish the physiological effect of afirst hormone receptor in a host from other hormone receptors in saidhost which respond to the same ligand, said method comprising:

replacing the ligand binding domain of said first receptor with a ligandbinding domain from an exogenous receptor to produce a chimeric receptormaintained under the control of a tissue specific promoter;

wherein said exogenous receptor and the ligand to which the exogenousreceptor responds are not normally present in said host; and

wherein said exogenous receptor, in the presence of its associatedligand, binds to a hormone response element, thereby activating saidresponse element, and thereafter

monitoring the production of product(s) whose expression is controlledby said first hormone receptor when said host is exposed toultraspiracle receptor and ligand to which said exogenous receptorresponds.

In accordance with yet another embodiment of the present invention,there is provided a method to render a mammalian hormone receptoruniquely responsive to a ligand not endogenous to host(s) in which saidreceptor is normally found, said method comprising:

replacing the ligand binding domain of said receptor with a ligandbinding domain from a second receptor;

wherein said second receptor is not normally present in said host; andwherein the ligand to which the second receptor responds is not normallypresent in said host.

In accordance with a still further embodiment of the present invention,there is provided a method to determine the ligand(s) to which orphanreceptor(s) responds, said method comprising:

monitoring a host cell containing a reporter construct and a hybridreceptor for expression of product(s) of said reporter construct uponcontacting said cell with potential ligands for said orphan receptor andthe ultraspiracle receptor;

wherein said reporter construct comprises a gene encoding a reportermolecule, operatively linked for transcription to a steroid orsteroid-like hormone response element; wherein said response element isnot normally present in the cells of said host;

wherein said hybrid receptor comprises:

the N-terminal domain and DNA binding domain of a member of thesteroid/thyroid superfamily of receptors, wherein said member is notnormally present in the host cells, and wherein said member, in thepresence of its associated ligand, binds said response element,activating transcription therefrom, and

the ligand binding domain of said orphan receptor.

In accordance with yet another embodiment of the present invention,there is provided an isolated DNA which encodes the ultraspiraclereceptor, as described above as well as functional fragments thereof.The complete nucleotide sequence for the ultraspiracle receptor ispresented in SEQ ID NO:1 [see also, Oro et al., in Nature 347:298-301(1990)]. Also contemplated within the scope of the present invention aresequences encoding polypeptides comprising a DNA binding domain withsubstantially the same sequence as that of amino acids 104-169 shown inSEQ ID NO:2 (i.e., the DNA binding domain of the ultraspiraclereceptor). Also contemplated are sequences encoding polypeptides havingsubstantially the same sequence as that of amino acids 1-513 shown inSEQ ID NO:2. Also contemplated are sequences having substantially thesame nucleotide sequence as nucleotides 163-1704 shown in SEQ ID NO: 1.As employed herein, the term “substantially the same as” refers to DNAhaving at least about 70% homology with respect to the nucleotidesequence of the DNA fragment with which subject DNA is being compared.Preferably, DNA “substantially the same as” a comparative DNA will be atleast about 80% homologous to the comparative nucleotide sequence; withgreater than about 90% homology being especially preferred. Alsocontemplated are DNAs able to hybridize to the above-describedsequences, and having substantially the same functional propertiesthereof. A presently preferred DNA of the invention is the ECoRIfragment of vector pXR2C8 [see Oro, et al., supra).

DNA of the invention can optionally be incorporated into expressionvector(s) operative in a cell in culture to make the ultraspiraclereceptor (or functional fragments thereof) by expression of said DNA insaid cell. For example, the transcription of DNA can be controlled bythe Drosophila melanogaster actin 5C promoter. Host cells which canemployed for expression of said DNA include Drosophila melanogasterSchneider line 2 cells, Kc cells, and the like.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example I Plasmids

CMX-ECR was constructed by digesting pActEcR plasmid [Koelle et al.,Cell Vol. 67:59-77 (1991)] with HindIII. The resulting HindIII fragment,which contains the EcR coding region, was then inserted into CMXPL1, aderivative of CMX expression vector [Umesono et al. Cell Vol.65:1255-1266 (1991)]. Expression plasmid CMX-usp was made by insertingthe EcoRI fragment from the cDNA clone [Oro et al., Nature Vol.347:298-301 (1990)] which contains all the usp coding region into CMXPL1vector. ΔMTV-EcRE5CAT was constructed by ligation of an EcRE-containingoligonucleotide (SEQ ID NO:22):

5′-AGCTCGATGG ACAAGTGCAT TGAACCCTTG A       GCTACC TGTTCACGTA ACTTGGGAAC TTCGA

into HindIII-cleaved ΔMTV-CAT (Hollenberg and Evans, Cell Vol.55:899-906 (1988)). Restriction analysis and sequencing of the constructindicated that it contains 5 copies of this oligonucleotide.

GEcR was constructed by ligation of a NotI/BamHI fragment containing theDNA and hormone binding domains of a modified EcR cDNA, EcRnx, in placeof the DNA and hormone binding domains of the similarly modifiedglucocorticoid receptor expression construct pRShGRnx [Giguere et al.,Nature Vol. 330:624-629 (1987)]. The modified receptor CDNA wasconstructed using the site-directed mutagenesis procedure of Kunkel,T.A., Proc. Natl. Acad. Sci. USA Vol. 82:448-492 (1985) to insert NotI(employing SEQ ID NO:23 as the oligonucleotide template) and XhoI(employing SEQ ID NO:24 as the oligonucleotide template) sitesimmediately flanking the DNA binding domain. SEQ ID NO:23 is:

5′-CCTGCGCCAC GGCGGCCGCC GGAGCTGTG CCTG; and SEQ ID NO:24 is:5′-GTGGGTATG CGCCTCGAGT GCGTCGTCCC.

This mutagenesis procedure results in conversion of amino acids 258-260from ValGlnGlu to ArgProPro and amino acid 331 from Pro to Leu.

Example II Preparation of receptor protein. cell extracts and gelmobility shift assay

To generate protein in vitro, suitable plasmids for human (h)RARα, hTRB,hVDR, rat PPAR, Drosophila (d)usp and dEcR were linearized withrestriction enzyme 3′ of the termination codon. The linearized templateswere used for in vitro transcription and then translation using rabbitreticulocyte lysate according to manufacturer's instruction (Promega).Drosophila embryo extract was a gift from Dr. J. Kadonaga and preparedas described by Zoeller et al., in Genes Dev., Vol. 2:68-81 (1988).Schneider cell extracts were prepared following the procedures in Dammet al., Nature Vol. 339:593-597 (1989) and Umesono et al. (1991), supra.The extraction buffer contained 0.4 M KCl in 20 mM HEPES pH 7.5, 20%glycerol, 2 mM DTT and 1 mM PMSF.

For gel mobility shift assay, proteins were incubated with bindingbuffer, which contained 100 mM KCl, 7.5% glycerol, 20 mM HEPES pH 7.5, 2mM DTT and 0.1% NP-40, on ice for 20 minutes in the presence of 2 μg ofnonspecific competitor poly dI-dC and other oligo competitors. Thenapproximately 1 ng of ³²P-dCTP probe, which was labelled to specificactivity about 1-5×10⁸ cpm/μg by fill-in reaction with Klenow fragments,was added to the reaction and incubated at room temperature for 20minutes. Antiserum or preimmune serum was added 10 minutes after theprobe was added. The reaction was then loaded into 5% non-denaturingpolyacrylamide gel in 0.5×TBE running buffer (1×TBE comprises 0.089 MTris borate, 0.089 M boric acid and 0.002 M EDTA). Afterelectrophoresis, the gel was dried for autoradiography.

Example III Preparation of usp antiserum

Primers were designed to amplify the usp coding region which eithercovered the entire N-terminal and DNA binding domain (from amino acid 1to 210; GST-uspN) or the complete coding region (GST-usp) by polymerasechain reaction (PCR). The amplified fragments were subcloned into PGEX2Tvector (Pharmacia) for expression in bacteria. The expression of GSTfusion protein was performed according to the manufacturer's directions(Pharmacia).

The fusion protein GST-uspN was prepared and fractionated on SDSpolyacrylamide gel and the band corresponding to the fusion protein wasexcised. The gel slice was fragmented and used to immunize the rabbit atthree week intervals. The rabbit sera were collected and tested byWestern Blot for the ability to recognize usp protein. The positive serawere further purified by the procedure described in Vaughan et al., Met.in Enzymol. (Academic Press Vol. 168:588-617 (1989) with slightmodification. Briefly, full length GST-usp fusion protein and GSTprotein were purified using Glutathione Sepharose 4B (Pharmacia). Thepurified proteins were individually coupled to affi-gel 10 according tothe manufacturer's protocol (Biorad).

To affinity purify the antibody, the crude antiserum was first incubatedwith GST-coupled affi-gel for 2.5 hours at 4° C. with gentle rocking.The unbound fraction was separated from the beads by centrifugation. Thesupernatant was then incubated with full length GST-usp coupled affi-gelovernight at 4° C. with gentle rocking. The contents were then packedinto column and washed with 50 mM HEPES pH 7.5 supplemented with 0.5 MNaCl. The bound antibodies were eluted by 100 mM glycine. The elutedfractions were neutralized with 1 M Tris pH 8 and pooled, then dialyzedagainst PBS buffer which contained 0.02% of sodium azide. The purifiedantiserum was concentrated by Centricon 30 (Amicon) before it wasstored. This purified antibody is very specific as it does not crossreact with RXRs. It also does not react with other closely related flynuclear receptors including seven up (svp) type I, II [Mlodzik et al.,Cell 60:211-224 (1990)).

Example IV Cotransfection assay

Transfection was performed with calcium-phosphate precipitation methodas described previously [Umesono et al., Nature 336:262-264 (1989)]. CV1monkey kidney cells were maintained in Dulbeccots modified Eagle'smedium (DMEM) supplemented with 10% calf bovine serum. The cells weretransfected for 8-9 hours and then the DNA precipitates were washed awayand replaced with fresh medium with 10% charcoal-resin double strippedfetal bovine serum. 20-hydroxy-ecdysone (Sigma; 10 mg/ml in ethanol), orethanol alone was then given to the cells. 24-28 hours later, the cellswere harvested. Beta-galactosidase (βGal) activity was measured and anormalized amount of extract was used for CAT assay (Umesono et al.(1989) supra]. The following amount of plasmid DNA was included in the10 cm plate transfection: 250 ng each of CMX-EcR and RSV-GEcR; 500 ng ofCMX-usp; 5 μg ΔMTV-EcRE₅-CTA; 5 μg of βGal internal control plasmidCH111 (a derivative of CH110, Promega). The amount of CMX plasmid waskept constant in each transfection by adding CMX-luciferase. PGEM4 wasadded to bring the total amount of plasmid DNA to 15 μg per plate.

EXAMPLE V usp is the Drosophila nuclear factor that can enhance RAR DNAbinding activity

It has previously been shown that the DNA binding activity ofbacterially expressed RAR can be enhanced by adding cell extracts to thebinding reaction (Yang et al., Proc. Natl. Acad. Sci. USA 88:3559-3563(1991)). Extracts prepared from both mammalian cells and the DrosophilaSchneider cell line 2 (S2) had similar effects (Yang et al., supra]. Thepresence of this enhancing activity in Drosophila cells indicates that ageneral conserved mechanism may be utilized in both mammals andDrosophila to regulate DNA binding activity of receptors like RAR. Toaddress this question, experiments were set up to characterize thisDrosophila nuclear activity in S2 cell extracts and in embryo extracts.

In gel mobility shift assays using a ³²P-labelled natural RAR responseelement—βRARE [see SEQ ID NO: 15, see also de Thé et al., in Nature Vol.343:177-180 (1990); and Sucov et al., in Proc. Natl. Acad. Sci. USA Vol.87:5392-5398 (1990)] as the probe, in vitro translated RARα wasincubated with probe under binding conditions either alone or with 2 μgof S2 extracts or embryo extracts. In vitro translated RARα, by itself,did not bind with appreciable affinity. For mammalian extracts [Glass etal., Cell Vol. 63:729-738 (1990)], incubating the RARα with either S2 orDrosophila embryo extracts dramatically enhanced DNA binding activity,while cell extract alone did not show similar binding activity.

Two clues suggested the possibility that the observed enhancing activitymight be the ultraspiracle receptor (usp). First, RXR, the putativevertebrate homologue of usp, has been shown to enhance RAR DNA binding[Yu et al., Cell Vol. 67:1251-1266 (1991); Kliewer et al., Nature Vol.355:446-449 (1992); Leid et al., Cell Vol. 68:377-395 (1992)]. Second,usp protein has been found to be present in both S2 and embryo extractswith relative abundance. Based on these observations, it wasinvestigated whether usp is the Drosophila nuclear activity that caninteract with RAR. An affinity purified antibody against usp, preparedas described in Example III, was added to the mobility shift reaction.The affinity purified antibody supershifted the majority of the proteinDNA complex while preimmune serum had no effect in the mobility pattern.By incubating with higher concentration of usp antibody, essentially allthe binding complex was supershifted. These results indicate that theactivity present in both types of fly extracts can be attributed to uspprotein and that usp is likely the major factor in the extract whichinteracts with RAR.

To further show that usp is indeed the Drosophila component involved inthe RAR interactions, usp protein was in vitro translated in the rabbitreticulocyte lysate (see Example II for procedure employed) and testedwhether in vitro translated usp can mimic the fly extracts' activity tointeract with RARα. Neither in vitro translated usp alone, nor RARαalone, bound to a βRARE probe. However, when the two proteins wereincubated together, a prominent retarded complex appeared. This complexcomigrated with the complex detected in the RAR and cell extract mixingexperiments. The presence of both usp and RAR protein in thisprotein/DNA complex was demonstrated by the antibodies raised againstusp and RARα. Either the affinity purified usp antibody, or the RARα,specifically affected the protein/DNA complex, while preimmune sera hadno effect. This complex likely represents an RAR/usp heterodimer, as itsmobility is similar to the RXR/RAR complex which has been proposed to bea heterodimeric species [Yu et al., supra; Kliewer et al., supra; Leidet al., supra]. Thus it is concluded that usp is the Drosophila nuclearactivity which can interact with RAR in binding to specific RARE via theformation of a putative heterodimer.

Example VI usp can heterodimerize with several members of the mammaliannuclear receptor family

The finding that usp is able to heterodimerize with RAR suggested thatit would be appropriate to check whether this interaction reflected ageneral ability of usp to form heterodimers with other members of thesteroid/thyroid superfamily. Using in vitro translated usp protein,interaction of usp with three mammalian nuclear receptors (TRβ, VDR andPPAR) was tested in gel mobility shift assays with the appropriateresponse element for each of the three receptors as the probes. Responseelements used were as follows:

Response SEQ Element Abbreviation Sequence IN NO: AOX-PPARE (DR1)¹AGGACA   A   AGGTCA 25 SSP1-VDRE (DR3)² GGTTCA  CGA  GGTTCA  7 MLV-TRE(DR4)³ GGGTCA  TTTC AGGTCC 12 βRARE (DR5)⁴ GGTTCA CCGAA AGTTCA 15¹Kliewer et al., submitted to Nature entitled “9-Cis Retinoic Acid andPeroxisome Proliferator Signalling Pathways Converge Througn RXRα-PPARInteractions” ²Noda et al., Proc. Natl. Acad. Sci. USA 87:9995-9999(1990) ³Sap et al., Nature 340:242-244 (1989) ⁴Sucov et al., supra.

For a review providing further discussion with respect to these responseelements, see Umesono et al., 1991, supra. With any of the receptorsalone (prepared by in vitro translation from cDNA clones) there was verylittle or no binding to the test probes. However, when they wereincubated with usp, a dramatic increase in DNA binding activity could bedetected. In a usp dependent fashion, TRβ bound to a natural TR responseelement (MLV-TRE) and VDR/usp bound to SSP1-VDRE, a natural VDR responseelement. The usp and PPAR interaction was tested on a PPARE derived fromthe acyl CoA oxidase promoter (AOX, Kliewer et al., submitted, supra,and references cited therein). PPAR/usp complex bound to AOX-PPARE withhigh affinity. The usp antibody again showed the presence of usp inthose complexes by shifting the retarded bands in all threecombinations, whereas preimmune serum did not affect the bindingpattern. Oligonucleotide competition assay demonstrated that the uspdependent heterodimers all showed correct response element specificity.Therefore, by interacting with usp to form heterodimer, all fourmammalian receptors tested achieved high affinity DNA binding to theircognate response elements. It can be concluded, therefore, that theability of usp to interact with other receptors to form heterodimers isa characteristic feature of usp, and that receptor heterodimerformation, as exemplified by RXR and usp, is conserved betweenvertebrates and invertebrates.

Example VII Ecdvsone receptor and usp can heterodimerize to form a highaffinity DNA binding complex

If the substitution of usp for RXR in heterodimerization with RAR andother mammalian receptors represents a conserved feature of mammalianand Drosophila receptors, it can be speculated that there might be oneor more Drosophila activities which can interact with usp. The ligandbinding domain has been shown to contain the dimerization domain forsome nuclear receptors [Forman et al., Mol. Endocrinol. Vol. 3:1610-1626(1989); Fawell et al., Cell 60:953-962 (1990)] and it is also essentialfor interaction between RAR and nuclear factors including RXR (Glass etal., supra; Kliewer et al. supra). Sequence comparisons reveal that,with respect to the ligand binding domain, all RXR heterodimer partners,including RAR, TR and VDR, are much more similar to one another than toother receptors, particularly RXR. Among the Drosophila receptormembers, ecdysone receptor (EcR) is one of the receptors which showsstrong homology to RAR, VDR and TR within this region. This homologysuggests EcR may have an evolutionarily conserved domain that, like RAR,VDR and TR, allows EcR to interact with usp.

To test the potential interaction between usp and EcR, experiments werecarried out to determine whether usp is part of the defined EcR DNAbinding activity present in ecdysone responsive Schneider 2 cells[Koelle et al. Cell 67:59-77 (1991)]. As shown by Koelle et al., in agel mobility shift assay using a natural ecdysone response elementderived from hsp27 promoter (³² P-labelled hsp27-EcRE, Riddihough andPelham, EMBO J. Vol. 6:3729-3734 (1987)) as the probe, one specificmajor complex could be detected in the S2 extract. This complex can becompeted away by specific cold oligonucleotide but not by the unrelatedoligonucleotide competitor GREpal, a glucocorticoid response element. Todetermine whether usp is present in this EcRE binding complex, affinitypurified usp antibody was added to the reaction. usp antibody cansupershift the specific EcRE binding complex from the S2 extract but notthe lower minor complex, which was much less sensitive to the specificcold oligonucleotide competition. Preimmune serum had no effect on theupper major complex but it disrupted the lower minor complex. Antibodyagainst RXRα and RARλ did not affect the specific complex. Therefore,these data demonstrate that usp is part of the EcRE DNA binding complexpresent in S2 cells, strongly suggesting an interaction between EcR andusp.

The EcR and usp interaction was also tested under more definedconditions. In vitro translated EcR and usp were prepared and theirinteraction was assayed by gel mobility shift assays. Using the samehsp27-EcRE as the probe, usp did not bind to this element by itself. EcRalso failed to bind to hsp27-EcRE, in contrast to the EcRE bindingactivity in S2 cells. To test whether usp can complement EcR DNAbinding, as it does with mammalian receptors, both usp and EcR wereco-incubated in the reaction. In the presence of both receptors, a novelhigh affinity DNA binding complex appeared. Usp antibody, but notpreimmune serum, can supershift the complex, demonstrating that usp ispart of the complex. This complex is proposed to be a heterodimericspecies, which is consistent with the observation of other uspheterodimers. These data demonstrate that EcR binding to ecdysoneresponse element (hsp27-EcRE) depends upon usp and are consistent withthe existence of a functionally significant EcR/usp complex.

Example VIII DNA binding activity of EcR/usD heterodimer is correlatedwith the ecdysone responsiveness in vivo

To establish that the EcR/usp heterodimer is physiologically relevant,it was set out to determine whether the DNA binding properties ofEcR/usp heterodimer can be correlated with ecdysone responsiveness invivo. This was done by testing EcR/usp heterodimer binding to severalwild type and mutant EcREs characterized by their differential abilityto mediate the ecdysone responsiveness in cultured cells (for review seeCherbas et al., Genes Dev. Vol. 5:120-131 (1991)). The response elementsused in this study are set forth below. The position and the orientationof ERE-like half sites (AGGTCA-like) are marked by arrows. For example,the palindromic motif in hsp27-EcRE is indicated by arrows arranged as→←. The mutated nucleotides in 11N and 15N-EcRE are in small letters.The arrow in 11N-EcRE covers where the remaining palindromic motifextends. In Eip28/29-EcRE, which is named as dis*-Eip28/29 in Cherbas etal., supra, the half site which can constitute a highly degeneratedpalindrome is marked with a wavy, broken line. Note that two ERE halfsites are present in the configuration of direct repeats spaced by threenucleotides in Eip28/29-EcRE. The ability of individual responseelements to mediate ecdysone response in cultured cells (summarized fromCherbas et al., supra) and to serve as high affinity binding site forEcR/usp complex are summarized to the right of the sequences.

Ability to Ability to Function Mediate as Binding Site Ecdysone forECR/usp Response Multimeric Complex         -----> hsp27-EcREATTGGACAAGTGCATTGAACCCTTGTCTCT    +    + (SEQ ID NO:26)TAACCAGTTCACGTAACTTGGGAACAGAGA                <-----          ---->11N-EcRE     atgctGTGCATTGAACgtgctcga    −    − (SEQ ID NO:27)    tacgaCACGTAACTTGcacgagct                <----         ----->15N-EcRE     atgAAGTGCATTGAACCCgctcga    +    + (SEQ ID NO:28)    tacTTCACGTAACTTGGGcgagct                <-----    ˜˜˜˜˜˜>Eip28/29-EcRE TAAAGGATCTTGACCCCAATGAACTTCTTA    +    + (SEQ ID NO:29)ATTTCCTAGAACTGGGGTTACTTGAAGAAT           <-----   <-----

An EcRE derived from the Drosophila Eip28/29 gene has been shown tomediate ecdysone response in cultured cells [Cherbas et al., supra]. Incontrast to the hsp27-EcRE palindrome, the Eip28/29-EcRE is a compositeelement containing a direct repeat and a highly degenerated palindromicmotif. The ability of EcR/usp complex to recognize this EcRE wasexamined. This element can effectively compete the EcR/usp binding tohsp27-EcRE. This competition is as effective as that of the hsp27-EcREitself, demonstrating that the Eip28/29-EcRE is also a high affinitybinding site for EcR/usp complex. In contrast, an unrelated competitor(GREpal) had no effect on the DNA binding. The high affinity binding bythe EcR/usp complex parallels the ability the Eip28/29-EcRE to mediateecdysone response in culture cells.

In contrast to the high affinity binding referred to above, a mutanthsp27-EcRE (referred as 11-N-hsp by Cherbas et al., supra, wherein thetwo nucleotides at the ends of the palindrome and the flanking sequencewere changed), failed to serve as a high affinity binding site for theEcR/usp. This oligonucleotide did not compete the specific binding ofEcR/usp to hsp27-EcRE, which was consistent with the observation thatthis mutated EcRE failed to confer ecdysone responsiveness to aheterologous promoter in transfection assay (Cherbas et al., supra).However, a back mutation which regenerated the palindrome motif(15-N-hsp) led to recovery of the ecdysone responsiveness and effectivecompetition for the EcR/usp binding to the wild type hsp27-EcRE.Therefore, the ability of a specific EcRE motif to mediate the ecdysoneresponse in vivo correlated well with its ability to serve as highaffinity binding site for EcR/usp complex. These data suggest that theEcR/usp complex can mediate the ecdysone response in vivo.

Example IX usp is present in the embryonic ECRE binding activity

Phenotypic analysis of usp reveals that it is required at a number ofdevelopmental events which are correlated with known or potentialecdysone-regulated processes. For example, usp is found to be anecessary component for the completion of embryogenesis [Oro et al.,“The Drosophila retinoid X receptor homolog ultraspiracle functions inboth female reproduction and eye morphogenesis”, Development, in press(1992)]. The presence of the ecdysone pulse (Richards, G., Mol. andCell. Endocrinol. Vol. 21:181-197 (1981)) as well as the EcR protein(Koelle et al., supra) during embryogenesis indicates that EcR is alsorequired during embryonic development.

Based on the coexpression and functional requirement of both activities,it was next tested whether EcR and usp can interact during thisdevelopmental stage. To this end, the EcRE binding activities in theembryo extract were determined by mobility shift assay using 5-10 μg ofembryo extract and ³²P-labelled hsp27-EcRE as the probe. Specific EcREbinding complexes can be detected, as demonstrated by specificoligonucleotide and unrelated oligonucleotide competition. Indeed, uspis present in those complexes, as usp antibody was able to supershiftthose complexes. Preimmune serum did not alter the mobility pattern,although a slightly enhanced overall DNA binding was observed. Thedetection of multiple EcRE binding complexes is consistent with theexistence of multiple forms of EcR protein which have been reported(Koelle et al., supra). The identities of these complexes as EcRcomplexes were supported by the fact that the upper complex comigratedwith the EcR complex present in the S2 extract and that the DNA bindingspecificity of these complexes as assayed by oligonucleotide competitionwas indistinguishable from the EcR/usp complex prepared in vitro.

Based on these data, it can be concluded that the embryonic EcRE bindingcomplexes contain usp. These data suggest an interaction betweenendogenous usp and EcR in the Drosophila embryo, where both activitiesare required for embryonic development.

Example X usp is required for ecdvsone responsiveness in heterologouscultured cells

The in vitro DNA binding data suggests that usp is required for EcR highaffinity DNA binding. The detection of EcR/usp heterodimeric DNA bindingcomplex from embryo implies that they may interact in vivo. To determinewhether usp and EcR can functionally interact in vivo, cotransfectionexperiments were set up to assay if usp is required for EcR to exert anecdysone response in cultured cells. Mammalian cells were chosen as theheterologous system because they do not contain endogenous EcR and uspbackground.

Ecdysone response in CV1 cells was determined by using anecdysone-responsive reporter gene (ΔMTV-EcRE₅-CAT), which contains thesame core hsp27-EcRE motif tested in DNA binding assay describedearlier. The chloramphenicol acetyl transferase (CAT) activity inducedby ecdysone was measured in the presence of different combinations oftransfected EcR and usp expression vectors. CAT activity is expressed inFIG. 2 as the percentage of conversion which is normalized against thelevel in EcR alone at the presence of ecdysone assigned as one (column1, shaded rectangle). The final concentration of 20-hydroxy-ecdysone(Sigma) is 40 μM. Columns 1 and 2 show that transfection with EcR alone(column 1) or usp alone (Column 2) does not respond to 20-OH-ecdysonetreatment. Cotransfection of EcR and usp together is shown in column 3and the induction of CAT activity could be observed by about 3 fold. Therequirement of EcRE for induction is demonstrated by using the parentalΔMTV-CAT as reporter. This construct does not respo nd to ecdysone(column 4).

As shown in FIG. 2A, transfection of EcR alone failed to conferresponsiveness to 20-hydroxy-ecdysone, consistent with the idea that EcRby itself cannot mediate ecdysone response. To test whether usp cancomplement the EcR activity as it did in the DNA binding assay, uspexpression vector was cotransfected with EcR into CV1 cells.Cotransfection of usp with EcR indeed was able to confer a significantresponse to 20-OH-ecdysone (over 3 fold, FIG. 2A). This induction wasstrictly dependent on both EcR and EcRE, as transfection with uspexpression plasmid alone or a reporter construct with out EcRE(ΔMTV-CAT) did not result in ecdysone responsiveness (FIG. 2A). Thelevel of ind uction by EcR and usp in CV1 cells is significant butsomewhat lower than expected compared with the interaction in DNAbinding assay.

To address the possi bility that EcR may not function properly inmammalian cells, the N-terminal domain of the glucocorticoid receptor(GR) was fused to the EcR DNA binding domain and ligand binding domainto create the construct GEcR (see Example 1). The presence of the GRN-terminal domain, which contains a transactivating domain [Hollenbergand Evans, supra], should increase the transcriptional potency andprovide an optimal translation signal for Drosophila EcR protein tofunction properly in th e mammal ia n cells. This construct retained theability to interact with usp in DNA binding assay. Since usp did notinteract with GR in either DNA binding or in transfection assay, it isclear that GR N-terminal fusion should not affect the basic property ofEcR to interact with usp. A similar GR fusion protein with TR has beenshown to behave like wild type TR except that it is a more potenttransactivator [Thompson and Evans, Proc. Natl. Acad. Sci. USA Vol.86:3494-3498 (1989)]. Therefore this system should increase thesensitivity of a functional interaction between usp and EcR whilefaithfully retaining the basic property of EcR.

As shown in FIG. 2B, expression plasmid of GEcR was transfected into CV1cells alone, or with usp expression plasmid. The conversion of CAT wasnormalized as described for panel A. Note that the scales of the CATconversion in FIG. 2, panels A and B are different. Similar to theresults obtained with wild type EcR, transfection of GEcR alone failedto confer ecdysone response. However, when cotransfected with the uspexpression plasmid, the 20-OH-ecdysone treatment induced CAT activity by8-10 fold (FIG. 2B). This induction was also dependent on the presenceof the EcRE in the reporter constructs. Thus, the GEcR mediated ecdysoneresponse is similar to EcR except the signal level is higher (compareFIGS. 2A and 2B). In conclusion, the presence of usp appears to beessential for EcR and GEcR to exert an ecdysone response in CV1 cells.These data demonstrate that EcR and usp can interact in vivo andconstitute a functional ecdysone response in a heterologous cell line.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

29 2304 base pairs nucleic acid single linear DNA (genomic) not providedCDS 163..1701 1 GGACACGGTG GCGTTGGCAA AGTGAAACCC CAACAGAGAG GCGAAAGCGAGCCAAGACAC 60 ACCACATACA CACGAAGAGA ACGAGCAAGA AGAAACCGGT AGGCGGAGGAGGCGCTGCCC 120 CCAGTTCCTC CAATATACCC AGCACCACAT CACAAGCCCA GG ATG GACAAC TGC 174 Met Asp Asn Cys 1 GAC CAG GAC GCC AGC TTT CGG CTG AGC CACATC AAG GAG GAG GTC AAG 222 Asp Gln Asp Ala Ser Phe Arg Leu Ser His IleLys Glu Glu Val Lys 5 10 15 20 CCG GAC ATC TCG CAG CTG AAC GAC AGC AACAAC AGC AGC TTT TCG CCC 270 Pro Asp Ile Ser Gln Leu Asn Asp Ser Asn AsnSer Ser Phe Ser Pro 25 30 35 AAG GCC GAG AGT CCC GTG CCC TTC ATG CAG GCCATG TCC ATG GTC CAC 318 Lys Ala Glu Ser Pro Val Pro Phe Met Gln Ala MetSer Met Val His 40 45 50 GTG CTG CCC GGC TCC AAC TCC GCC AGC TCC AAC AACAAC AGC GCT GGA 366 Val Leu Pro Gly Ser Asn Ser Ala Ser Ser Asn Asn AsnSer Ala Gly 55 60 65 GAT GCC CAA ATG GCG CAG GCG CCC AAT TCG GCT GGA GGCTCT GCC GCC 414 Asp Ala Gln Met Ala Gln Ala Pro Asn Ser Ala Gly Gly SerAla Ala 70 75 80 GCT GCA GTC CAG CAG CAG TAT CCG CCT AAC CAT CCG CTG AGCGGC AGC 462 Ala Ala Val Gln Gln Gln Tyr Pro Pro Asn His Pro Leu Ser GlySer 85 90 95 100 AAG CAC CTC TGC TCT ATT TGC GGG GAT CGG GCC AGT GGC AAGCAC TAC 510 Lys His Leu Cys Ser Ile Cys Gly Asp Arg Ala Ser Gly Lys HisTyr 105 110 115 GGC GTG TAC AGC TGT GAG GGC TGC AAG GGC TTC TTT AAA CGCACA GTG 558 Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg ThrVal 120 125 130 CGC AAG GAT CTC ACA TAC GCT TGC AGG GAG AAC CGC AAC TGCATC ATA 606 Arg Lys Asp Leu Thr Tyr Ala Cys Arg Glu Asn Arg Asn Cys IleIle 135 140 145 GAC AAG CGG CAG AGG AAC CGC TGC CAG TAC TGC CGC TAC CAGAAG TGC 654 Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr Gln LysCys 150 155 160 CTA ACC TGC GGC ATG AAG CGC GAA GCG GTC CAG GAG GAG CGTCAA CGC 702 Leu Thr Cys Gly Met Lys Arg Glu Ala Val Gln Glu Glu Arg GlnArg 165 170 175 180 GGC GCC CGC AAT GCG GCG GGT AGG CTC AGC GCC AGC GGAGGC GGC AGT 750 Gly Ala Arg Asn Ala Ala Gly Arg Leu Ser Ala Ser Gly GlyGly Ser 185 190 195 AGC GGT CCA GGT TCG GTA GGC GGA TCC AGC TCT CAA GGCGGA GGA GGA 798 Ser Gly Pro Gly Ser Val Gly Gly Ser Ser Ser Gln Gly GlyGly Gly 200 205 210 GGA GGC GGC GTT TCT GGC GGA ATG GGC AGC GGC AAC GGTTCT GAT GAC 846 Gly Gly Gly Val Ser Gly Gly Met Gly Ser Gly Asn Gly SerAsp Asp 215 220 225 TTC ATG ACC AAT AGC GTG TCC AGG GAT TTC TCG ATC GAGCGC ATC ATA 894 Phe Met Thr Asn Ser Val Ser Arg Asp Phe Ser Ile Glu ArgIle Ile 230 235 240 GAG GCC GAG CAG CGA GCG GAG ACC CAA TGC GGC GAT CGTGCA CTG ACG 942 Glu Ala Glu Gln Arg Ala Glu Thr Gln Cys Gly Asp Arg AlaLeu Thr 245 250 255 260 TTC CTG CGC GTT GGT CCC TAT TCC ACA GTC CAG CCGGAC TAC AAG GGT 990 Phe Leu Arg Val Gly Pro Tyr Ser Thr Val Gln Pro AspTyr Lys Gly 265 270 275 GCC GTG TCG GCC CTG TGC CAA GTG GTC AAC AAA CAGCTC TTC CAG ATG 1038 Ala Val Ser Ala Leu Cys Gln Val Val Asn Lys Gln LeuPhe Gln Met 280 285 290 GTC GAA TAC GCG CGC ATG ATG CCG CAC TTT GCC CAGGTG CCG CTG GAC 1086 Val Glu Tyr Ala Arg Met Met Pro His Phe Ala Gln ValPro Leu Asp 295 300 305 GAC CAG GTG ATT CTG CTG AAA GCC GCT TGG ATC GAGCTG CTC ATT GCG 1134 Asp Gln Val Ile Leu Leu Lys Ala Ala Trp Ile Glu LeuLeu Ile Ala 310 315 320 AAC GTG GCC TGG TGC AGC ATC GTT TCG CTG GAT GACGGC GGT GCC GGC 1182 Asn Val Ala Trp Cys Ser Ile Val Ser Leu Asp Asp GlyGly Ala Gly 325 330 335 340 GGC GGG GGC GGT GGA CTA GGC CAC GAT GGC TCCTTT GAG CGA CGA TCA 1230 Gly Gly Gly Gly Gly Leu Gly His Asp Gly Ser PheGlu Arg Arg Ser 345 350 355 CCG GGC CTT CAG CCC CAG CAG CTG TTC CTC AACCAG AGC TTC TCG TAC 1278 Pro Gly Leu Gln Pro Gln Gln Leu Phe Leu Asn GlnSer Phe Ser Tyr 360 365 370 CAT CGC AAC AGT GCG ATC AAA GCC GGT GTG TCAGCC ATC TTC GAC CGC 1326 His Arg Asn Ser Ala Ile Lys Ala Gly Val Ser AlaIle Phe Asp Arg 375 380 385 ATA TTG TCG GAG CTG AGT GTA AAG ATG AAG CGGCTG AAT CTC GAC CGA 1374 Ile Leu Ser Glu Leu Ser Val Lys Met Lys Arg LeuAsn Leu Asp Arg 390 395 400 CGC GAG CTG TCC TGC TTG AAG GCC ATC ATA CTGTAC AAC CCG GAC ATA 1422 Arg Glu Leu Ser Cys Leu Lys Ala Ile Ile Leu TyrAsn Pro Asp Ile 405 410 415 420 CGC GGG ATC AAG AGC CGG GCG GAG ATC GAGATG TGC CGC GAG AAG GTG 1470 Arg Gly Ile Lys Ser Arg Ala Glu Ile Glu MetCys Arg Glu Lys Val 425 430 435 TAC GCT TGC CTG GAC GAG CAC TGC CGC CTGGAA CAT CCG GGC GAC GAT 1518 Tyr Ala Cys Leu Asp Glu His Cys Arg Leu GluHis Pro Gly Asp Asp 440 445 450 GGA CGC TTT GCG CAA CTG CTG CTG CGT CTGCGC CGC TTT GCG ATC GAT 1566 Gly Arg Phe Ala Gln Leu Leu Leu Arg Leu ArgArg Phe Ala Ile Asp 455 460 465 CAG CCT GAA GTG CCA GGA TCA CCT GTT CCTCTT CCG CAT TAC CAG CGA 1614 Gln Pro Glu Val Pro Gly Ser Pro Val Pro LeuPro His Tyr Gln Arg 470 475 480 CCG GCC GCT GGA GGA GCT CTT TCT CGA GCAGCT GGA GGC GCC GCC GCC 1662 Pro Ala Ala Gly Gly Ala Leu Ser Arg Ala AlaGly Gly Ala Ala Ala 485 490 495 500 ACC CGG CCT GGC GAT GAA ACT GGA GTAGGG TCC CGA CTC TAAAGTCGCC 1711 Thr Arg Pro Gly Asp Glu Thr Gly Val GlySer Arg Leu 505 510 CCCGTTCTCC ATCCGAAAAA TGTTTCATTG TGATTGCGTTTGTTTGCATT TCTCCTCTCT 1771 ATCCCTACAA AAGCCCCCTA ATATTACGCA AAATGTGTATGTAATTGTTT ATTTTTTTTT 1831 TATTACCTAA TATTATTATT ATTATTGATA TAGAAAATGTTTTCCTTAAG ATGAAGATTA 1891 GCCTCCTCGA CGTTTATGTC CCAGTAAACG AAAAACAAACAAAATCCAAA ACTTGAAAAG 1951 AACACAAAAC ACGAACGAGA AAATGCACAC AAGCAAAGTAAAAGTAAAAG TTAAACTAAA 2011 GCTAAACGAG TAAAGATATT AAAATAACGG TTAAAATTAATGCATAGTTA TGATCTACAG 2071 ACGTATGTAA ACATACAAAT TCAGCATAAA TATATATGTCAGCAGGCGCA TATCTGCGGT 2131 GCTGGCCCCG TTCTAAACCA ATTGTAATTA CTTTTTAACATAAATTTACC CAAAACGTTA 2191 TCAATTAGAT GCGAGATACA AAAATCACCG ACGAAAACCAACAAAATATA TCTATGTATA 2251 AAAAATATAA GCTGCATAAC AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAA 2304 513 amino acids amino acid linear protein notprovided 2 Met Asp Asn Cys Asp Gln Asp Ala Ser Phe Arg Leu Ser His IleLys 1 5 10 15 Glu Glu Val Lys Pro Asp Ile Ser Gln Leu Asn Asp Ser AsnAsn Ser 20 25 30 Ser Phe Ser Pro Lys Ala Glu Ser Pro Val Pro Phe Met GlnAla Met 35 40 45 Ser Met Val His Val Leu Pro Gly Ser Asn Ser Ala Ser SerAsn Asn 50 55 60 Asn Ser Ala Gly Asp Ala Gln Met Ala Gln Ala Pro Asn SerAla Gly 65 70 75 80 Gly Ser Ala Ala Ala Ala Val Gln Gln Gln Tyr Pro ProAsn His Pro 85 90 95 Leu Ser Gly Ser Lys His Leu Cys Ser Ile Cys Gly AspArg Ala Ser 100 105 110 Gly Lys His Tyr Gly Val Tyr Ser Cys Glu Gly CysLys Gly Phe Phe 115 120 125 Lys Arg Thr Val Arg Lys Asp Leu Thr Tyr AlaCys Arg Glu Asn Arg 130 135 140 Asn Cys Ile Ile Asp Lys Arg Gln Arg AsnArg Cys Gln Tyr Cys Arg 145 150 155 160 Tyr Gln Lys Cys Leu Thr Cys GlyMet Lys Arg Glu Ala Val Gln Glu 165 170 175 Glu Arg Gln Arg Gly Ala ArgAsn Ala Ala Gly Arg Leu Ser Ala Ser 180 185 190 Gly Gly Gly Ser Ser GlyPro Gly Ser Val Gly Gly Ser Ser Ser Gln 195 200 205 Gly Gly Gly Gly GlyGly Gly Val Ser Gly Gly Met Gly Ser Gly Asn 210 215 220 Gly Ser Asp AspPhe Met Thr Asn Ser Val Ser Arg Asp Phe Ser Ile 225 230 235 240 Glu ArgIle Ile Glu Ala Glu Gln Arg Ala Glu Thr Gln Cys Gly Asp 245 250 255 ArgAla Leu Thr Phe Leu Arg Val Gly Pro Tyr Ser Thr Val Gln Pro 260 265 270Asp Tyr Lys Gly Ala Val Ser Ala Leu Cys Gln Val Val Asn Lys Gln 275 280285 Leu Phe Gln Met Val Glu Tyr Ala Arg Met Met Pro His Phe Ala Gln 290295 300 Val Pro Leu Asp Asp Gln Val Ile Leu Leu Lys Ala Ala Trp Ile Glu305 310 315 320 Leu Leu Ile Ala Asn Val Ala Trp Cys Ser Ile Val Ser LeuAsp Asp 325 330 335 Gly Gly Ala Gly Gly Gly Gly Gly Gly Leu Gly His AspGly Ser Phe 340 345 350 Glu Arg Arg Ser Pro Gly Leu Gln Pro Gln Gln LeuPhe Leu Asn Gln 355 360 365 Ser Phe Ser Tyr His Arg Asn Ser Ala Ile LysAla Gly Val Ser Ala 370 375 380 Ile Phe Asp Arg Ile Leu Ser Glu Leu SerVal Lys Met Lys Arg Leu 385 390 395 400 Asn Leu Asp Arg Arg Glu Leu SerCys Leu Lys Ala Ile Ile Leu Tyr 405 410 415 Asn Pro Asp Ile Arg Gly IleLys Ser Arg Ala Glu Ile Glu Met Cys 420 425 430 Arg Glu Lys Val Tyr AlaCys Leu Asp Glu His Cys Arg Leu Glu His 435 440 445 Pro Gly Asp Asp GlyArg Phe Ala Gln Leu Leu Leu Arg Leu Arg Arg 450 455 460 Phe Ala Ile AspGln Pro Glu Val Pro Gly Ser Pro Val Pro Leu Pro 465 470 475 480 His TyrGln Arg Pro Ala Ala Gly Gly Ala Leu Ser Arg Ala Ala Gly 485 490 495 GlyAla Ala Ala Thr Arg Pro Gly Asp Glu Thr Gly Val Gly Ser Arg 500 505 510Leu 71 amino acids amino acid unknown unknown peptide not provided 3 CysXaa Xaa Cys Xaa Xaa Asp Xaa Ala Xaa Gly Xaa Tyr Xaa Xaa Xaa 1 5 10 15Xaa Cys Xaa Xaa Cys Lys Xaa Phe Phe Xaa Arg Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45Xaa Xaa Xaa Lys Xaa Xaa Arg Xaa Xaa Cys Xaa Xaa Cys Arg Xaa Xaa 50 55 60Lys Cys Xaa Xaa Xaa Gly Met 65 70 15 base pairs nucleic acid singlelinear not provided 4 AGGTCAAGGA GGTCA 15 15 base pairs nucleic acidsingle linear not provided 5 GGGTGAATGA GGACA 15 15 base pairs nucleicacid single linear not provided 6 GGGTGAACGG GGGCA 15 15 base pairsnucleic acid single linear not provided 7 GGTTCACGAG GTTCA 15 16 basepairs nucleic acid single linear not provided 8 AGGTCACAGG AGGTCA 16 16base pairs nucleic acid single linear not provided 9 AGGTGACAGG AGGTCA16 16 base pairs nucleic acid single linear not provided 10 AGGTGACAGGAGGACA 16 16 base pairs nucleic acid single linear not provided 11GGGTTAGGGG AGGACA 16 16 base pairs nucleic acid single linear notprovided 12 GGGTCATTTC AGGTCC 16 17 base pairs nucleic acid singlelinear not provided 13 AGGTCACCAG GAGGTCA 17 17 base pairs nucleic acidsingle linear not provided 14 AGGTGAACAG GAGGTCA 17 17 base pairsnucleic acid single linear not provided 15 GGTTCACCGA AAGTTCA 17 17 basepairs nucleic acid single linear not provided 16 GGTTCACCGA AAGTTCA 1717 base pairs nucleic acid single linear not provided 17 AGGTCACTGACAGGGCA 17 17 base pairs nucleic acid single linear not provided 18GGGTCATTCA GAGTTCA 17 34 base pairs nucleic acid single linear notprovided 19 AAGCTTAAGG GTTCACCGAA AGTTCACTCA GCTT 34 38 base pairsnucleic acid single linear not provided 20 AAGCTTAAGG GTTCACCGAAAGTTCACTCG CATAGCTT 38 43 base pairs nucleic acid single linear notprovided 21 AAGCTTAAGG GTTCACCGAA AGTTCACTCG CATATATTAG CTT 43 62 basepairs nucleic acid single unknown not provided 22 AGCTCGATGG ACAAGTGCATTGAACCCTTG AGCTACCTGT TCACGTAACT TGGGAACTTC 60 GA 62 33 base pairsnucleic acid single unknown not provided 23 CCTGCGCCAC GGCGGCCGCCGGAGCTGTGC CTG 33 29 base pairs nucleic acid single unknown not provided24 GTGGGTATGC GCCTCGAGTG CGTCGTCCC 29 13 base pairs nucleic acid singleunknown not provided 25 AGGACAAAGG TCA 13 30 base pairs nucleic aciddouble unknown not provided 26 ATTGGACAAG TGCATTGAAC CCTTGTCTCT 30 24base pairs nucleic acid double unknown not provided 27 ATGCTGTGCATTGAACGTGC TCGA 24 24 base pairs nucleic acid double unknown notprovided 28 ATGAAGTGCA TTGAACCCGC TCGA 24 30 base pairs nucleic aciddouble unknown not provided 29 TAAAGGATCT TGACCCCAAT GAACTTCTTA 30

That which is claimed is:
 1. A method to indirectly induce or repress,in an in vitro expression system or in cells in culture, thetranscription activation of a first gene by a member of thesteroid/thyroid hormone superfamily of receptors which associates withat least the dimerization domain of ultraspiracle receptor, in thepresence of ligand for said member, wherein the expression of said geneis maintained under the control of a hormone response element to whichsaid member binds, said method comprising: exposing said expressionsystem to at least the dimerization domain of an ultraspiracle receptor,in an amount effective to form a multimeric complex receptor with saidmember.
 2. A method according to claim 1 wherein the dimerization domainof the ultraspiracle receptor is provided by exposing said system tocompound(s) and/or condition(s) which induce expression of a second geneencoding said dimerization domain.
 3. A method to directly or indirectlyinduce or repress, in an expression system, the transcription activationof a gene by a member of the steroid/thyroid superfamily of receptorswhich associates with at least the dimerization domain of ultraspiraclereceptor, in the presence of ligand for said member, and in the furtherpresence of at least the dimerization domain of an ultraspiraclereceptor, wherein the expression of said gene is maintained under thecontrol of a hormone response element to which said member is capable ofbinding, said method comprising: exposing said system to compound(s)and/or condition(s) which prevent association of said member with saiddimerization domain, to a degree effective to prevent said association.4. A method according to claim 4 wherein said compound which preventsassociation of said member with said ultraspiracle receptor is ananti-ultraspiracle antibody.
 5. A method for the expression of arecombinant product detrimental to a host organism, said methodcomprising: (a) transforming suitable host cells with: (i) a constructcomprising a sequence encoding said recombinant product under thecontrol of a hormone response element, wherein said hormone responseelement is not normally present in the cells of said host; and (ii) DNAencoding a member of the steroid/thyroid superfamily of receptors notnormally present in said host cells; (b) growing said host cells to thedesired level in the substantial absence of a hormone(s) which, incombination with said member, binds to said hormone response element;and (c) inducing expression of said recombinant product by introducinginto said host cells an ultraspiracle receptor and hormone(s) which, incombination with said member, bind to said hormone response element,thereby activating transcription therefrom.